1. Field of the Invention
Angiogenesis, the formation of blood vessels by sprouting from pre-existing ones, is essential for the growth of solid tumors beyond 2-3 mm in diameter and for tumor metastasis (Folkman, 1995; reviewed in Bouck et al., 1996;). The generation of new capillaries involves a multistep process, which includes the dissolution of the membrane of the originating vessel, the endothelial cell migration and proliferation, and formation of a new vascular tube (Cliff, 1963; Schoefl, 1963; Ausprunck and Folkman, 1977). Suppression of any one of these steps would inhibit the formation of new vessels and therefore affect tumor growth and generation of metastases. Indeed, it has been estimated that the elimination of a single endothelial cell could inhibit the growth of 100 tumor cells (Thorpe et al., 1995). Moreover, endothelial cells are genetically stable and therefore unlikely to mutate into drug-resistant variants (Young, 1989; Kerbel, 1991; Boehm et al., 1997). Since they line the inside of blood vessels, they are easily accessible to circulating drugs. This feature suggests that anti-angiogenic therapies targeting endothelial cells may provide a promising mechanism for cancer treatment.
2. Discussion of the Background
So far, several angiogenic factors have been identified (reviewed in Folkman, 1995; Hanahan et al., 1996), including the particularly potent Vascular Endothelial Growth Factor (VEGF), also known as VPF or vasculotropin (reviewed in Ferrara, 1993; Ferrara and Davis-Smyth, 1997). Unlike other angiogenic factors, VEGF acts as an endothelial cell-specific mitogen during angiogenesis (Terman et al., 1992 and Ferrara, 1993). Antibodies raised against VEGF have been shown to suppress tumor growth in vivo (Kim et al., 1993), indicating that VEGF antagonists could have therapeutic applications as inhibitors of tumor-induced angiogenesis.
VEGF was purified initially from the conditioned media of folliculostellate cells and from a variety of tumor cell lines (Ferrara et al, 1989; Plouxc3xat et al., 1989; Myoken et al., 1991). It is a member of the cystine-knot family of growth factors, which also includes PDGF (Platelet Derived Growth Factor). Recently, a number of VEGF structural homologs have been identified: VEGF-B, VEGF-C, VEGF-D and Placenta Growth Factor (PlGF) (Klagsbrun and D""Amore, 1996; reviewed in Ferrara, 1999). The human gene encoding VEGF is organized into eight exons, separated by seven introns. Alternative splicing of mRNAs for the VEGF gene results in the generation of five different molecular species, having 121, 145, 165, 189, or 206 amino acid residues in the mature monomer (Tisher et al., 1991; Houck et al., 1991). Only VEGF165, which lacks the residues encoded by exon 6, is the mature and active form of VEGF. It binds to heparin and cell surface heparan sulfate proteoglycans, and can be expressed as a free or as a cell membrane bound form (Houck et al., 1992). Two tyrosine kinase receptors have been identified for which VEGF acts as a high affinity ligand: a fins-like tyrosine kinase-1 (Fit-1 or VEGFR-1) and a kinase domain receptor (KDR/Flk-1 or VEGFR-2) (Matthews et al., 1991; Terman et al., 1991; De Vries et al., 1992; Millauer et al., 1993). Although Flt-1 binds VEGF with 50-fold higher affinity than KDR (De Vries et al., 1992), most of the VEGF angiogenic properties (mitogenicity, chemotaxis, and induction on morphological changes) are mediated by interaction with KDR (Waltenberger et al., 1994). Therefore, the interaction between VEGF and KDR is the most appropriate to interrupt in order to inhibit angiogenesis .
The screening of phage-displayed libraries is a powerful technique for identifying peptides mimicking protein surfaces (Smith, 1985; Hoess, 1993; Felici et al., 1995). Since each peptide is physically linked to a genetic particle, clones specifically binding a target molecule can be selected by consecutive cycles of in vitro biopanning and in vivo amplification. New agonists and antagonists for cell membrane receptors have been successfully identified using this process (Cwirla et al., 1990; Cortese et al., 1996), for example, RGD containing peptides that bind either the GPIIb/IIIa receptor on platelets (O""Neil et al., 1992) or the 5 1 integrin (Koivunen et al., 1993). The selected peptides were able to antagonize integrin-mediated cell adhesion.
The present inventors have identified peptides blocking the binding of VEGF to KDR. A random peptide library displayed on filamentous phages (Cortese et al., 1996) was screened using two parallel strategies. In the first, the peptide repertoire was screened with cells expressing recombinant KDR (Plouxc3xat et al., 1997) and in the second, with a monoclonal antibody raised against VEGF. Since this antibody blocked VEGF-dependent endothelial cell proliferation, we postulated that its antigen binding site mimics all or part of the VEGF interaction surface with KDR. Both strategies led to the isolation of peptides that compete with VEGF binding to KDR, including a peptide, ATWLPPR (SEQ ID NO:1), which specifically inhibited human endothelial cell proliferation in vitro. Moreover, it totally abolished VEGF-induced angiogenesis in vivo. ATWLPPR (SEQ ID NO:1), as a specific antagonist of VEGF-KDR interaction, may represent an effective anti-tumor agent.
Accordingly, it is one object of the present invention to provide a method for screening for peptides capable of interacting with VEGF.
It is another object of the present invention to provide novel peptides which inhibit the interaction of VEGF and KDR.
It is another object of the present invention to provide novel polynucleotide sequences which encode such peptides.
It is another object of this invention to provide vectors which comprise the polynucleotides encoding such peptides.
It is another object of this invention to provide methods of inhibiting angiogenesis and diseases affected by angiogenesis using such peptides.
It is another object of this invention to provide pharmaceutical compositions containing such peptides.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors"" discovery of the novel peptides disclosed herein.